TY - JOUR
T1 - Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry
AU - Mukherjee, Prithvijit
AU - Berns, Eric J.
AU - Patino, Cesar A.
AU - Hakim Moully, Elamar
AU - Chang, Lingqian
AU - Nathamgari, S. Shiva P.
AU - Kessler, John A.
AU - Mrksich, Milan
AU - Espinosa, Horacio D.
N1 - Funding Information:
Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health (NIH) under Award Number U54CA199091 and by NIH R21 Award Number GM132709‐01 and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T32GM105538. This work utilized the Argonne National Lab Center for Nanoscale Materials. Use of the Center for Nanoscale Materials, an Office of Science user facility, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE‐AC02‐06CH11357. Cells were obtained from the Northwestern University Developmental Therapeutics Core generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. This work made use of the EPIC facility of Northwestern University's NUANCE Center, which received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS‐1542205); the MRSEC program (NSF DMR‐1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN.
Funding Information:
Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health (NIH) under Award Number U54CA199091 and by NIH R21 Award Number GM132709-01 and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T32GM105538. This work utilized the Argonne National Lab Center for Nanoscale Materials. Use of the Center for Nanoscale Materials, an Office of Science user facility, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Cells were obtained from the Northwestern University Developmental Therapeutics Core generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. This work made use of the EPIC facility of Northwestern University's NUANCE Center, which received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN.
Publisher Copyright:
© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2020/7/1
Y1 - 2020/7/1
N2 - Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.
AB - Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.
KW - cell sampling
KW - electroporation
KW - enzyme activities
KW - microfluidics
KW - nanotechnologies
UR - http://www.scopus.com/inward/record.url?scp=85085557422&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85085557422&partnerID=8YFLogxK
U2 - 10.1002/smll.202000584
DO - 10.1002/smll.202000584
M3 - Article
C2 - 32452612
AN - SCOPUS:85085557422
SN - 1613-6810
VL - 16
JO - Small
JF - Small
IS - 26
M1 - 2000584
ER -